Reagents Supplied

Advantages and Features

Applications

*Ideally, DNA for transformation should be purified and resuspended in water or TE. However, up to 10 µl of DNA directly from a ligation mix can be used with only a two-fold loss of transformation efficiency. Where it is necessary to maximize the number of transformants (e.g. a library), a purification step, either a spin column or phenol/chloroform extraction and ethanol precipitation should be added.
Figure 1, vtPA activity assayed from crude lysates: Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt+ cells are DHB4, which is the parent of FÅ113 (Origami™).
Figure 2, PfCHT1 chitinase activity assayed from crude lysates: Plasmodium falciparum chitinase (PfCHT1) with three cysteines were expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.

Properties and Usage

Antibiotics for Plasmid Selection

Working Concentration

Ampicillin

100 μg/ml

Carbenicillin

100 μg/ml

Chloramphenicol

33 μg/ml

Kanamycin

30 μg/ml

Streptomycin

25 μg/ml

Tetracycline

15 μg/ml

Storage Temperature

-80°C

Antibiotic Resistance

str

spec

Notes

STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.

Usage Guidelines & Tips

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.

Functional Test (Disulfide Bond Formation):Cells are transformed with a plasmid expressing MBP-NucA fusion protein. When expressed at 37° C in E. coli, NucA is toxic to cells only in its oxidized disulfide-bonded state.

Transformation Efficiency:The competent cells are tested for transformation efficiency and pass minimum release criteria. Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 μg of plasmid into a given volume of competent cells.

Safety Data Sheet

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Research Use Only

This product is sold for research use only and not for resale in any form. Commercial use of this product may require a license. For license information, please contact the Licensing Office, New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938.

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